Cancer is one of the most deadly threats to human health. In the U.S. alone, cancer affects nearly 1.3 million new patients each year, and is the second leading cause of death after cardiovascular disease, accounting for approximately 1 in 4 deaths. Solid tumors are responsible for most of those deaths. Although there have been significant advances in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved only by about 10% in the past 20 years. Cancers, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making treatment extremely difficult.
CD33 is a 67 kDa transmembrane cell surface glycoprotein receptor. CD33 is a member of sialic acid-binding immunoglobulin-like lectins (SIGLEC) family. Proteins in this family mediate adhesion of leukocytes to endothelial cells by binding sialylated glycans. (Kelm S, Schauer R, Crocker P R. Glycoconj J. 1996; 13:913-926). In addition, CD33 functions as an inhibitory receptor through immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD33 receptor activation leads to phosphorylation of two tyrosines (Y340 and Y358) in CD33 cytoplasmic tail, which serves as a docking site for SHP phosphatases, and is involved in inhibitory signal transduction cascades, such as downregulation of calcium mobilization (Paul S P1, Taylor L S, Stansbury E K, McVicar D W Blood. 2000 Jul. 15; 96(2):483-90).
CD33 is a myeloid lineage differentiation antigen, and it is highly expressed on myeloid progenitor cells (Andrews R G, Torok-Storb B, Bernstein I D. Blood. 1983; 62:124-132), but is only expressed at low levels in differentiated myeloid cells, namely macrophages and granulocytes (Simmons D, Seed B. J Immunol. 1988; 141:2797-2800). By contrast, CD33 has been reported to be expressed on 87.8%-99% of acute myeloblastic leukemias (AML) (A Ehningerl et al. Blood Cancer Journal (2014) 4, e218; Christina Krupka et al. Blood 2014 123:356-365). AML is a devastating disease, with 5-year survival rate of approximately 26% (available on the world wide web at cancer.net/cancer-types/leukemia-acute-myeloid-aml/statistics). The present standard of care for AML consists of remission induction treatment by high dose of chemotherapy or radiation, followed by consolidation, comprised of allogeneic stem cell transplantation and additional courses of chemotherapy as needed (available on the world wide web at cancer.org/cancer/acute-myeloid-leukemia/treating/typical-treatment-of-aml.html). High toxicity associated with this treatment, as well as the risk of complications, such as myelosuppression or GVHD, motivate the search for better therapeutic alternatives.
A number of novel approaches to treat AML, including antibody-drug conjugates (SGN-CD33A, Vadastuximab Talirine, Stein A. S. et al. (2015). Blood, 126(23), 324; Phase I-II clinical trial NCT02706899), a bispecific T-cell-engaging antibody (AMG330, Laszlo G S et al. Blood 2013:123(4):554-561, NCT02520427), and CART-33 cells (Wang Q S et al. Mol Ther. 2015 January; 23(1):184-91, NCT01864902) are currently being investigated. However, several of the novel approaches have been held back due to clinical toxicity. Seattle Genetics Phase I clinical trials testing SGN-CD33 drug were recently put on hold due to risk of hepatotoxicity (available on the world wide web at businesswire.com/news/home/20161227005087/en/Seattle-Genetics-Announces-Clinical-Hold-Phase-1). Gemtuzumab ozogamicin (Mylotarg, Pfizer/Wyeth) was voluntarily withdrawn from the market by the manufacturer in 2010, following incidence of potentially fatal veno-occlusive liver disease observed in a post-marketing clinical trial (Jacob M. Rowe and Bob Löwenberg Blood 2013 121:4838-4841). Despite recent reintroduction of Mylotarg by FDA for CD33+ adult AML, and for relapsed/refractory pediatric AML, new, more conservative lower dosage and new regiments have been prescribed for this drug (FDA press release September 2017, available on the world wide web at fda.gov). The efficacy of this treatment, the durability of patients' responses to Mylotarg, instances of tumor antigen escape and its safety profile under the new regiments remain to be determined. Therefore, the need for safe efficacious and durable treatments for AML remains imminent.
Chimeric Antigen Receptors (CARs) are hybrid molecules comprising three essential units: (1) an extracellular antigen-binding motif, (2) linking/transmembrane motifs, and (3) intracellular T-cell signaling motifs (Long A H, Haso W M, Orentas R J. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2 (4):e23621). The antigen-binding motif of a CAR is commonly fashioned after an single chain Fragment variable (ScFv), the minimal binding domain of an immunoglobulin (Ig) molecule. Alternate antigen-binding motifs, such as receptor ligands (i.e., IL-13 has been engineered to bind tumor expressed IL-13 receptor), intact immune receptors, library-derived peptides, and innate immune system effector molecules (such as NKG2D) also have been engineered. Alternate cell targets for CAR expression (such as NK or gamma-delta T cells) are also under development (Brown C E et al Clin Cancer Res. 2012; 18(8):2199-209; Lehner M et al. PLoS One. 2012; 7 (2):e31210). There remains significant work with regard to defining the most active T-cell population to transduce with CAR vectors, determining the optimal culture and expansion techniques, and defining the molecular details of the CAR protein structure itself.
The linking motifs of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or designed to be an extended flexible linker. Structural motifs, such as those derived from IgG constant domains, can be used to extend the ScFv binding domain away from the T-cell plasma membrane surface. This may be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (such as for the disialoganglioside GD2; Orentas et al., unpublished observations). To date, the signaling motifs used in CARs always include the CD3-ζ chain because this core motif is the key signal for T cell activation. The first reported second-generation CARs featured CD28 signaling domains and the CD28 transmembrane sequence. This motif was used in third-generation CARs containing CD137 (4-1BB) signaling motifs as well (Zhao Y et al J Immunol. 2009; 183 (9): 5563-74). With the advent of new technology, the activation of T cells with beads linked to anti-CD3 and anti-CD28 antibody, and the presence of the canonical “signal 2” from CD28 was no longer required to be encoded by the CAR itself. Using bead activation, third-generation vectors were found to be not superior to second-generation vectors in in vitro assays, and they provided no clear benefit over second-generation vectors in mouse models of leukemia (Haso W, Lee D W, Shah N N, Stetler-Stevenson M, Yuan C M, Pastan I H, Dimitrov D S, Morgan R A, FitzGerald D J, Barrett D M, Wayne A S, Mackall C L, Orentas R J. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, Blood. 2013; 121 (7): 1165-74; Kochenderfer J N et al. Blood. 2012; 119 (12): 2709-20). This is borne out by the clinical success of CD19-specific CARS that are in a second generation CD28/CD3-ζ (Lee D W et al. American Society of Hematology Annual Meeting. New Orleans, La.; Dec. 7-10, 2013) and a CD137/CD3-signaling format (Porter D L et al. N Engl J Med. 2011; 365 (8): 725-33). In addition to CD137, other tumor necrosis factor receptor superfamily members such as OX40 also are able to provide important persistence signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009; 15(18):5852-60). Equally important are the culture conditions under which the CAR T-cell populations were cultured.
Current challenges in the more widespread and effective adaptation of CAR therapy for cancer relate to a paucity of compelling targets. Creating binders to cell surface antigens is now readily achievable, but discovering a cell surface antigen that is specific for tumor while sparing normal tissues remains a formidable challenge. One potential way to imbue greater target cell specificity to CAR-expressing T cells is to use combinatorial CAR approaches. In one system, the CD3-ζ and CD28 signal units are split between two different CAR constructs expressed in the same cell; in another, two CARs are expressed in the same T cell, but one has a lower affinity and thus requires the alternate CAR to be engaged first for full activity of the second (Lanitis E et al. Cancer Immunol Res. 2013; 1(1):43-53; Kloss C C et al. Nat Biotechnol. 2013; 31(1):71-5). A second challenge for the generation of a single ScFv-based CAR as an immunotherapeutic agent is tumor cell heterogeneity. At least one group has developed a CAR strategy for glioblastoma whereby the effector cell population targets multiple antigens (HER2, IL-13Ra, EphA2) at the same time in the hope of avoiding the outgrowth of target antigen-negative populations. (Hegde M et al. Mol Ther. 2013; 21(11):2087-101).
T-cell-based immunotherapy has become a new frontier in synthetic biology; multiple promoters and gene products are envisioned to steer these highly potent cells to the tumor microenvironment, where T cells can both evade negative regulatory signals and mediate effective tumor killing. The elimination of unwanted T cells through the drug-induced dimerization of inducible caspase 9 constructs with AP1903 demonstrates one way in which a powerful switch that can control T-cell populations can be initiated pharmacologically (Di Stasi A et al. N Engl J Med. 2011; 365(18):1673-83). The creation of effector T-cell populations that are immune to the negative regulatory effects of transforming growth factor-β by the expression of a decoy receptor further demonstrates that degree to which effector T cells can be engineered for optimal antitumor activity (Foster A E et al. J Immunother. 2008; 31(5):500-5). Thus, while it appears that CARs can trigger T-cell activation in a manner similar to an endogenous T-cell receptor, a major impediment to the clinical application of this technology to date has been limited in vivo expansion of CAR+ T cells, rapid disappearance of the cells after infusion, and disappointing clinical activity. A number of antibody-based modalities targeting CD33-positive tumors are currently in development, including an anti-CD33 antibody-drug conjugate (Stein A. S. et al. Blood, 2015, 126(23), 324), a bispecific T cell engager (BiTE), (Laszlo G S et al. Blood 2013:123(4):554-561), and CART cells (Wang Q S et al. Mol Ther. 2015 January; 23(1):184-91). Recent work in pre-clinical models of AML has shown that lysis of CD33 positive AML blasts and tumor cell lines by CD33-targeting modalities can be achieved in vitro and in vivo, however a number of challenges to this approach became apparent in the clinical context, including treatment-associated toxicity (available on the world wide web at businesswire.com/news/home/20161227005087/en/Seattle-Genetics-Announces-Clinical-Hold-Phase-1; Rowe J M and Löwenberg B, Blood 2013 121:4838-4841, Wang Q S et al. Mol Ther. 2015 January; 23(1):184-91, NCT01864902) and suboptimal efficacy, (Walter R B, et al. Blood. 2012; 119(26): 6198-6208; Cowan A J, et al. Biosci 2013; 18(4):1311-1334). Moreover, in BiTEs-based approach, the reliance upon high-density CD33 antigen expression and the need for additional T cell co-stimulation/checkpoint blockage for optimal BiTE function remain a challenge (Laszlo G S et al. Blood. 2014; 123(4):554-56, Laszlo G S et al. Blood Cancer Journal (2015) 5, e340). Accordingly, there is an urgent and long felt need in the art for discovering novel compositions and methods for treatment of AML using an approach that can exhibit specific and efficacious anti-tumor effect without the aforementioned short comings.
The present invention addresses these needs by providing CAR compositions and therapeutic methods that can be used to treat cancers and other diseases and/or conditions. In particular, the present invention as disclosed and described herein provides CARs that may be used the treatment of diseases, disorders or conditions associated with dysregulated expression of CD33 and which CARs contain CD33 antigen binding domains that exhibit a high surface expression on transduced T cells, exhibit a high degree of cytolysis and transduced T cell in vivo expansion and persistence.